RESEARCH PAPER |
Regulation of betaine synthesis by precursor supply and choline monooxygenase expression in Amaranthus tricolor
1Research Institute, Meijo University, Nagoya, 468-8502, Japan
2Graduate School of Environmental and Human Sciences, Meijo University, Nagoya, 468-8502, Japan
* To whom correspondence should be addressed. E-mail: takabe{at}ccmfs.meijo-u.ac.jp
Received 18 June 2007; Revised 15 October 2007 Accepted 18 October 2007
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Abstract |
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In plants, betaine is synthesized upon abiotic stress via choline oxidation, in which choline monooxygenase (CMO) is a key enzyme. Although it had been thought that betaine synthesis is well regulated to protect abiotic stress, it is shown here that an exogenous supply of precursors such as choline, serine, and glycine in the betaine-accumulating plant Amaranthus tricolor further enhances the accumulation of betaine under salt stress, but not under normal conditions. Addition of isonicotinic acid hydrazide, an inhibitor of glycine decarboxylase, inhibited the salinity-induced accumulation of betaine. Salt-induced accumulation of A. tricolor CMO (AmCMO) and betaine was much slower in roots than in leaves, and a transient accumulation of proline was observed in the roots. Antisense expression of AmCMO mRNA suppressed the salt-induced accumulation of AmCMO and betaine, but increased the level of choline
2– 3-fold. This indicates that betaine synthesis is highly regulated by AmCMO expression. The genomic DNA, including the upstream region (1.6 kbp), of AmCMO was isolated. Deletion analysis of the AmCMO promoter region revealed that the 410 bp fragment upstream of the translation start codon contains the sequence responsive to salt stress. These data reveal that the promoter sequence of CMO, in addition to precursor supply, is important for the accumulation of betaine in the betaine-accumulating plant A. tricolor. Key words: Abiotic stress, Amaranthus, betaine, choline monooxygenase, glycine, salt stress, serine
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Introduction |
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Many bacteria, plants, and animals accumulate glycine betaine (GB), hereafter betaine, under water or salt stress conditions. In these organisms, betaine is synthesized through two steps of choline oxidation, choline
betaine aldehydebetaine (Rathinasabapathi et al., 1997; Takabe et al., 2006). The second step is catalysed by NAD+-dependent betaine aldehyde dehydrogenase (BADH) in plants, animals, and bacteria. Different enzymes are involved in the first step in different organisms. In plants, a novel Rieske-type iron–sulphur enzyme, choline monooxygenase (CMO), catalyses this step (Burnet et al., 1995; Rathinasabapathi et al., 1997), whereas membrane-bound choline dehydrogenase (CDH) or soluble choline oxidase (COX) catalyses the first step in animals and bacteria (Takabe et al., 2006). Thus far, CMO has only been found in Chenopodiaceae and Amaranthaceae, but not in some betaine-accumulating plants such as the mangrove (Russel et al., 1998; Hibino et al., 2001). Since many crop plants do not have a betaine synthetic pathway, genetic engineering of betaine biosynthesis pathways represents a potential way to improve crop plant stress tolerance (Chen and Murata, 2002; Hanson and Gregory, 2002). Choline oxidation enzymes such as CMO, CDH, and COX have been introduced into non-betaine-accumulating plants, and this has often improved stress tolerance. However, the engineered levels of betaine are generally low, and the increases in tolerance commensurately small (Hibino et al., 2002; Park et al., 2004). Flux analysis suggests that the import of choline into chloroplasts limits the level of betaine (Nuccio et al., 2000). Subsequent works showed that increasing the supply of choline or ethanolamine increased betaine levels (Nuccio et al., 1998; McNeil et al., 2001). Moreover, the level of phosphatidylethanolamine methyltransferase is 100-fold higher in betaine-accumulating plants than in non-betaine-accumulating plants (Nuccio et al., 2000). These facts suggest that the enzymes involved in betaine synthesis are well regulated to protect against harmful stresses.
Regulation of betaine synthesis is important, but its mechanisms are poorly understood. This is partly due to the difficulty associated with transforming betaine-accumulating plants such as spinach, barley, and mangrove. Using the betaine-accumulating C4 plant, Amaranthus tricolor, it is shown that an exogenous supply of precursors for betaine (glycine, serine, and ethanolamine) increases the betaine level even in this betaine-accumulating plant. A transient assay system was also developed for elucidating the functional roles of betaine synthetic genes and used to demonstrate that AmCMO is a key player in betaine synthesis. Moreover, the genomic DNA including the promoter of AmCMO was isolated, and it was shown that a 410 bp region upstream of the translation start codon of AmCMO contains the sequence responsive to salt stress.
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Materials and methods |
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Plant materials
Amaranthus tricolor was used throughout this study. For expression of the upstream region of the AmCMO gene, Arabidopsis thaliana ecotype Columbia (Col-0) was used to construct transgenic plants. Growth of A. tricolor and A. thaliana was carried out as described previously (Wang et al., 1999; Waditee et al., 2005).
Stress treatments
One-month-old A. tricolor plants grown hydroponically were used for stress treatment. For salinity stress, different concentrations of NaCl were applied to the liquid culture medium with 1/10 MS solutions. For dehydration stress, leaves were kept without solution for 12 h. For other stresses, detached leaves were collected from 1-month-old plants, and various stresses [methyl viologen, 20 µM; Fe, 100 µM; H2O2, 100 µM; abscisic acid (ABA), 50 µM; cold, 4 °C] were applied with liquid 1/10 MS solution on a Petri dish for 24 h.
Quantitative RT-PCR and western blotting
Quantification of AmCMO mRNA expression was carried out with a TaqMan fluorescent chemical analysis method (Waditee et al., 2002). Total RNA was isolated from the leaves of young seedlings by the phenol–chloroform method. First-strand cDNA syntheses were performed using reverse transcriptase (Stratagene, La Jolla, CA, USA). The open reading frame of AmCMO was amplified from first-strand cDNA using gene-specific primers. The synthesis of TaqMan fluorescent probe, the Taq probe, was performed by Perkin-Elmer Japan. The clone-specific primer was used for amplification. A 50 µl aliquot of the reaction mixture was used, containing PCR products of 10 ng of total RNA, 1x TaqMan buffer A, 5.5 µl of MgCl2, 300 µmol l–1 dATP, dGTP, and dCTP, 600 µmol l–1 dUTP, 0.2 µmol l–1 forward and reverse primers, 0.1 µmol l–1 TaqMan probe, and 1.25 U of Taq Gold. The PCR conditions were 15 s at 95 °C and 1 min at 60 °C. A previously described computer algorithm was used for quantification of AmCMO mRNA (Waditee et al., 2002).
SDS–PAGE and western blot analyses were carried out according to the standard protocol as described previously (Waditee et al., 2005). An antiserum raised against the spinach CMO was prepared using a white New Zealand female rabbit as previously described (Hibino et al., 2002). Protein contents were determined by the Lowry method (Hibino et al., 2002).
Analysis of betaine and choline
Betaine and choline was extracted as described previously (Waditee et al., 2005). After esterification, betaine and choline were measured by time of flight mass spectroscopy (KOMPACT MALDI TOF-MS, Shimadzu/Kratos) using d11-betaine and d9-choline, respectively, as internal standards.
Construction of antisense AmCMO
The targeted fragment of AmCMO for antisense expression was amplified from first-strand cDNA using AmCMOantBam660-F1 and AmCMOantSac-R1 primers. The sequences of all the primers are shown in Table 1. The PCR fragment was digested with BamHI and SacI, and ligated into the corresponding sites of the pBI101H.35S binary vector to generate the construct pBI101H.35S:antiCMO. This construct was transformed into Agrobacterium tumefaciens strain LBA4404 by electroporation. The positive clone was selected on medium containing kanamycin and hygromycin (50 mg l–1) and used for transient assay.
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Construction of binary vectors containing different upstream regions fused to a GUS gene
The 5'-flanking region of the AmCMO gene was amplified from genomic DNA using AmCMOproHind-F1 and AmCMOproNco-R1 primers. The amplified fragment was ligated into the EcoRV site of pBluescript II SK+ to generate pBSK:AmCMOpro. Three forward primers (AmCMOproHind-F2, AmCMOproHind-F3, and AmCMOproHind-F4 for
1240, 820, and 410, respectively) and reverse primers were used to amplify different deleted upstream regions from the pBSK:AmCMOpro plasmid. Four fragments were ligated into the HindIII- and NcoI-digested sites of the pCAMBIA1301 binary vector where they fused with a β-glucuronidase (GUS) reporter gene. These constructs were transformed into A. tumefaciens strain LBA4404 by electroporation, and positive clones were selected by growing on LB agar medium containing hygromycin (50 mg l–1).
Agro-infiltration of leaf transient assay and construction of transgenic Arabidopsis plants
Agrobacterium-mediated transient transformation was conducted as described previously (Yang et al., 2002). Fully expanded young leaves were used for agro-infiltration. For stable transformation of Arabidopsis, the floral dip method (Clough and Bent, 1998) was used.
GUS staining and activity
Quantitative GUS enzyme assays were performed as described by Jefferson et al. (1987). Briefly, pooled tissues were ground in GUS extraction buffer [100 mM sodium phosphate, pH 7.0, 10 mM EDTA, 0.1% (v/v) Triton X-100, 0.1% (w/v) SDS, and 10 mM 2-mercaptoethanol]. After centrifugation, protein concentrations in the supernatant were determined by the Lowry method. To reduce background activity, the extracts were heated at 55 °C for 30 min. The extracts (20 µg of protein) were incubated with 4-methylumbelliferyl glucuronide solution (0.5 mM) for 60 min at 37 °C. The reaction was stopped by addition of sodium carbonate. Fluorescence (excitation at 365 nm and emission at 455 nm) was measured with a Shimadzu RF-5300PC spectrofluorophotometer. GUS activity was calibrated with 4-methylumbelliferone.
GUS staining was carried out as previously described (Jefferson et al., 1987). Stress-treated or non-treated seedlings were immersed immediately in GUS staining solution containing 0.25 mM 5-bromo-4-chloro-3-indolyl β-D-glucuronide cyclohexylammonium salt in 50 mM sodium phosphate buffer (pH 7.3), and then incubated at 37 °C for 20 h. Stained plants were washed with ethanol.
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Results |
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Effects of salt on the accumulation levels of CMO and betaine in A. tricolor
Amaranthus tricolor plants were grown for 1 month and then 0.3 M NaCl was applied. Figure 1A shows that before salt stress, the levels of betaine and CMO were very low or almost undetectable, whereas significant levels of BADH were detected. Upon salt stress with 0.3 M NaCl for 12 h, the levels of CMO and betaine increased significantly, whereas the level of BADH increased only moderately. Upon removal of NaCl stress, both CMO and BADH decreased to the original levels within 72 h, although betaine remained at half the maximum level (Fig. 1A). It was also observed that the levels of mRNA for AmCMO increased upon salt stress and decreased upon release of salt stress (data not shown). Figure 1B shows the effects of different concentrations of NaCl on the accumulation of AmCMO and betaine. The levels of AmCMO and betaine were highly induced at 48 h after the addition of 0.1 M and 0.3 M NaCl, but their induction was low in response to 0.5 M NaCl owing to the severe stress of A. tricolor.
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In addition to salt stress, various other stresses were also examined. Expression of AmCMO was also induced by drought and ABA stresses (data not shown). Application of H2O2 stress slightly induced the expression of AmCMO, while other stresses such as methylviologen, Cu2+, Hg+, and high temperature did not induce the expression of AmCMO (data not shown). In contrast, expression of BADH was not stimulated by any of these stresses except methylviologen, which decreased the expression of AmCMO. Like CMO, the level of betaine was also enhanced by drought and ABA. These results indicate that the expression of AmCMO, but not BADH, is closely linked to the accumulation of betaine in A. tricolor.
Supply of precursors enhanced the level of betaine in A. tricolor
Recent studies on genetic engineering of betaine synthesis indicate that choline supply is important for the accumulation of betaine in non-betaine-accumulating plants in which the gene coding for the choline oxidation enzyme was introduced (Nuccio et al., 1998, 2000; Hibino et al., 2002). Therefore, it was of interest to examine whether the limitations of choline occur in betaine-accumulating A. tricolor. Amaranthus tricolor leaves have three colours: green, red, and yellow. Chlorophyll contents in yellow and red colour regions are very low, about 10% that of green colour regions, although photosynthetic activity is about 40% of that of green colour regions (Wang et al., 1999). As shown in Fig. 2A, the levels of choline in 1-month-old A. tricolor leaves were low among the three colour regions. Upon treatment with 0.3 M NaCl, the level of choline was increased in all colour regions and was saturated 7 d after the addition of NaCl. Addition of 1 mM choline slightly increased the level of choline. The level of betaine was also increased upon salt stress and was saturated 3 d after salt stress treatment (Fig. 2B). Inclusion of 1 mM choline significantly increased the betaine level to about 50 µmol g–1 FW 12 d after the addition of NaCl. Figure 2C shows the effects of an exogenous supply of various precursors on the levels of betaine and choline 3 d after salt treatment. Addition of choline, ethanolamine, glycine, and serine essentially did not affect the level of choline, but significantly increased the level of betaine. These results indicate that the in vivo supply of betaine precursor in A. tricolor was insufficient under salt stress conditions, but was adequate under normal conditions.
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Effects of isonicotinic acid hydrazide and CO2 on the accumulation of betaine
Since glycine and serine are precursors for betaine synthesis, the effects of isonicotinic acid hydrazide (INH) on the levels of betaine in A. tricolor were examined. INH is an inhibitor of the glycine decarboxylase complex, which is involved in glycine metabolism and/or glycine to serine conversion. As shown in Fig. 3A, INH decreased the salt-induced accumulation of betaine. The supply of serine increased the betaine level in salt-stressed A. tricolor to 20 µmol g–1 FW, but INH inhibited this level to 13 µmol g–1 FW. In contrast, the levels of choline were not altered significantly as much upon INH addition. These results indicate that glycine decarboxylase is at least partly involved in betaine accumulation.
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In photosynthetic tissues, glycine and serine are produced by photorespiration, the activity of which would depend on the ratio between oxygenation and carboxylation reactions of the RuBisCO enzyme. Therefore, the effects of CO2 concentration on the levels of betaine under salt stress conditions were examined. It was anticipated that the increase in the carboxylation reaction of RuBisCO by high CO2 would decrease the levels of precursors, such as glycine, serine, ethanolamine, and choline, leading to decreased levels of betaine. In support of this, Fig. 3B shows that the levels of betaine were low under conditions of high CO2 and high salinity.
Differential expression of AmCMO and betaine in sink and source organs
Next, the salt-induced expression of AmCMO and betaine in sink and source organs was examined. As shown in Fig. 4A, the level of betaine in the leaves increased after 6 h of salt, reaching a level of 13 µmol g–1 FW after 24 h of salt stress. A modest increase in the level of betaine was observed in the root. The level of CMO after salt stress for 24 h was much more pronounced in the leaves than in the roots (Fig. 4B). Enhanced levels of CMO in the leaves and the roots upon salt stress correlated well with the betaine levels observed in leaves and roots. Interestingly, the level of proline in the leaves was low during salt stress, whereas a rapid increase in the level of proline was observed in the roots (Fig. 4A). Figure 4C shows that intracellular levels of Na+ increased more rapidly in the roots than in the leaves, whereas K+ remained relatively unchanged in both tissues. The expression levels of AmCMO as well as the levels of betaine were much higher in young leaves compared with old ones (Fig. 4A, D). These results indicate that expression of AmCMO and betaine is higher in source organs than in sink organs.
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Antisense expression of AmCMO suppressed the protein level and reduced the accumulation of betaine at high salinity
To date, transformation of A. tricolor has not been reported. Despite extensive trials for transformation, it was not possibble to generate transgenic A. tricolor, but it was possible to express foreign genes in A. tricolor. As a result, a transient assay for AmCMO in the leaves was examined. Agrobacterium cells harbouring control and antisense AmCMO vectors were infiltrated into the leaf (Fig. 5A). After 24 h under normal conditions, low-level expression of AmCMO in both control and antisense AmCMO vector leaves was observed (Fig. 5B). However, when plants were treated with 0.3 M NaCl for 24 h after infiltration, AmCMO expression was highly induced in control vector leaves, but was significantly reduced in the antisense AmCMO leaves (Fig. 5B). A similar expression pattern was observed for the accumulation of betaine (Fig. 5C). Upon initiation of salt stress, accumulation of betaine was significantly increased in control vector leaves, but only slightly increased in the antisense AmCMO leaves. Choline increased concomitantly with betaine in control vector leaves during salt stress, but increased significantly more in the antisense AmCMO leaves (Fig. 5C). These results indicate that the infiltrated antisense AmCMO vector was transcribed in cells and inhibited the translation of AmCMO. Consequently, choline oxidation and betaine synthesis were decreased, resulting in the over-accumulation of choline in antisense AmCMO leaves.
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Isolation and characterization of the AmCMO promoter
The results described above show that the accumulation of betaine was induced by precursors (choline, ethanolamine, glycine, and serine), salt, drought, and low CO2. The levels of betaine correlated well with the levels of AmCMO. Therefore, it was of interest to analyse the promoter region of AmCMO. For this purpose, the genomic DNA of AmCMO was sequenced by chromosomal walking. As shown in Fig. 6A, the total length of genomic AmCMO is
10 831 bp (accession number AB303388). It contains 10 exons and nine introns. There is a long intron between the first and second exons. Excluding the first and last exons, the exons are short in length. The open reading frame of the AmCMO gene (accession number AB303389) is 1326 bp and encodes a protein of 442 amino acids that showed high similarity to spinach CMO deduced from cDNA (Rathinasabapathi et al., 1997) (data not shown).
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To characterize the promoter of AmCMO,
1.65 kb of the 5'-flanking region of the AmCMO gene was sequenced (Fig. 6). Numerous potential cis-acting elements were predicted by database analysis (PLACE; http://www.dna.affrc.go.jp/PLACE/) (data not shown). Besides the TATA-box and CAAT-box, several stress-related transcription-binding motifs were found. The WRKY transcription factor-binding sites (Eulgem et al., 1999) were found at –117, –237, –307, –994, and –1341. MYC-binding sites for rd22 (Abe et al., 1997) were found at –346 and –382. In addition, early responsive to dehydration (ERD1) (Simpson et al., 2003) sites were found at –396, –884, and –1349, abscisic acid-responsive-like elements (ABREs) (Nakashima et al., 2006) at –884 and –1349, and heat shock element (HSEs) (Rieping et al., 1992) at –828 and –1543. More cis-regulatory elements were found, although the roles of these putative elements in the transcriptional regulation of AmCMO were not tested.
Analysis of the AmCMO promoter by deletion of different upstream regions
To investigate the salt-responsive region of the AmCMO promoter, four kinds of deleted promoters fused to a GUS reporter gene were constructed (Fig. 7A). All four promoter–reporter constructs as well as an empty vector (without promoter) were transferred via the A. tumefaciens LBA4404 strain to A. tricolor leaves by infiltration. The infiltrated leaves were treated or not with 0.3 M NaCl for 24 h. The GUS activity assay revealed that the leaves infiltrated with empty vector did not show any activity (Fig. 7B). All four constructs containing various lengths of promoters showed relatively high activity under normal conditions. Under salt stress conditions, this activity increased. However, the 410 construct exhibited the highest activity, showing an 4-fold-increase upon salt stress. These results suggest that the –410 bp region contains the salt-inducible elements. Further identification of narrower promoter regions remains unsuccessful.
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Transgenic Arabidopsis plants expressing the
410 construct showed high GUS activity under conditions of high salinityThe above data showed that the –410 bp (D410) fragment of the promoter region contains the salinity response element. The importance of the D410 region was further tested in a heterologous system by generating transgenic Arabidopsis plant harbouring D410 fused to a GUS reporter gene. Several independent lines of transgenic plants were analysed to evaluate the promoter activity of the D410 fragment under salt-stress conditions. GUS staining of the salt-stressed plants was remarkably higher compared with non-stressed plants (Fig. 8A, B). GUS expression was mainly observed in vascular tissues. No expression was observed in the root tip. GUS activity was increased upon increasing the concentration of NaCl (Fig. 8C). Moreover, GUS activity was reduced about 20–30% by dark treatments (Fig. 8D). These results support the argument that the –410 fragment contains the region(s) responsive to salt and light.
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Discussion |
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Betaine is an important osmoprotectant and is synthesized upon abiotic stresses. Although regulation of betaine synthesis is an important subject, the molecular mechanisms remain poorly understood. In this study, the genomic DNA of AmCMO was sequenced. As shown in Fig. 6, it contains 10 exons and nine introns. The 5'-flanking region of the AmCMO gene,
1.65 kbp, was also sequenced. Numerous potential cis-acting elements were predicted by database analysis. Since Figs 2 and 3 demonstrate that the accumulation of betaine was induced by precursors (choline, ethanolamine, glycine, and serine), salt, drought, and low CO2, it is interesting to analyse these putative elements in the transcriptional regulation of AmCMO under stress conditions. From deletion analysis of promoter regions, it was demonstrated that the D410 fragment contains salt-inducible elements (Fig. 7). Expression of the D410 fragment in a heterologous species, A. thaliana, demonstrated that it contains the region(s) responsive to salt and light (Fig. 8). The presence of a light-dependent element is reasonable since reduced ferredoxin is required for the function of CMO (Brouquisse et al., 1989). In fact, the D410 fragment contains a ‘GT-1’ element (GAAAA) at –48 and an ‘I-box’ (GATA) at –283 and –363, which have been previously shown to be involved in NaCl- and light-regulated expression, respectively (Terzaghi et al., 1995; Park et al., 2002). Since all four fragments contain these two elements, one might expect that some other cis-acting elements in the upper region of the D410 fragment suppress their activation, and deletion of the upper regions exhibited the activity of ‘GT-1’ and ‘I-box’ elements in the D410 fragment. Due to the presence of other putative elements in the D410 fragment, further studies are needed to understand the interactions between these elements. Regardless of these facts, the present data indicate that the salt-inducible D410 fragment can be utilized as a salt-inducible promoter.
Figure 3A indicates that glycine decarboxylase activity is a limiting factor for the accumulation of betaine. In photosynthetic tissues, glycine decarboxylase is involved in photorespiration. The results presented in Fig. 3A suggest that upon salinity stress, glycine decarboxylase contributes to the accumulation of betaine by supplying the precursor substrates, serine or glycine. In non-photosynthetic tissues, the importance of serine synthesis via phosphorylated pathway from 3-phosphoglycerate has been reported (Ho et al., 1999). In addition to this, the importance of glycine decarboxylase and serine hydroxymethyltransferase for the metabolism of serine and glycine has also been reported (Hartung and Ratcliffe 2002). Thus, the role of glycine decarboxylase in betaine synthesis in non-photosynthetic tissues remains unclear. However, in photosynthetic tissues, the present data can be interpreted as follows. Abiotic stresses such as salt and drought often cause the closure of stomata, resulting in a decrease in cellular CO2. Subsequently, the ratio of carboxylation/oxygenation reactions would decrease, which, in turn, would increase the synthesis of glycine and serine via photorespiration for utilization in betaine synthesis. The precise interaction of glycine betaine synthesis and the photorespiratory pathway remains elusive.
Transformation of betaine-accumulating plants is an important step for understanding the molecular mechanism of betaine synthesis regulation. However, despite extensive trials for transformation, it was not possible to produce transgenic A. tricolor, but foreign genes could be expressed in A. tricolor using a transient leaf assay. Figure 5C shows that the antisense suppression of CMO expression significantly inhibited the salt-induced accumulation of betaine, whereas the level of choline increased 2– 3-fold (Fig. 5). This demonstrates a direct link between choline and betaine accumulation and their regulation by AmCMO expression. The transient assay approach used in this study could be useful as an alternative method to obtain transgenic plants for the elucidation of molecular mechanisms regulating betaine synthesis.
Figure 4A shows that the level of proline in leaves was low during salt stress, whereas a rapid increase in the level of proline was observed in the roots. Figure 4C shows that Na+ increased more rapidly in roots than in leaves, whereas K+ levels remained relatively unchanged in both leaf and root. The molecular mechanisms regulating these phenomena remain to be clarified. In addition, Fig. 4D shows that young leaves accumulated more betaine than old leaves. This might be due to the fact that CMO is slightly more abundant in young leaves. CMO expression in the roots of A. tricolor is not consistent with sugar beet, in which high expression was reported by Russell et al. (1998). The molecular mechanisms of these differences remain to be clarified.
Levels of accumulation of betaine in betaine-accumulating plants such as A. tricolor were often >30 µmol g–1 FW (Figs 1, 2). Introduction of betaine biosynthetic pathways into non-betaine-accumulating plants results in a low level accumulation of betaine. In the latter case, it was shown that choline import into chloroplasts and supply of precursors such as choline limit the levels of betaine (Nuccio et al., 1998, 2000). Even though exogenous choline was supplied, the betaine level remained low, <3 µmol g–1 FW. This indicates that an additional factor limits the accumulation of betaine. More extensive studies are needed to elucidate the mechanisms responsible for this effect. Although it has been thought that betaine synthesis is well regulated to protect against abiotic stresses, the data presented in this study clearly show that an exogenous supply of precursors such as choline, serine, and glycine in the betaine-accumulating plant, A. tricolor, enhances the accumulation of betaine under salt stress, but not under normal conditions. This suggests that betaine synthesis is not completely regulated, and it would therefore be possible to increase betaine levels in betaine-accumulating plants by genetic engineering of the precursor pathway for betaine synthesis. It would be interesting to examine whether these phenomena are also observed in other betaine-accumulating plants.
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Acknowledgements |
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We thank Eiko Tsunekawa for her expert technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and the High-Tech Research Center of Meijo University.
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Abbreviations |
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ABA, abscisic acid; ABRE, abscisic acid-responsive-like element; AmCMO, Amaranthus tricolor choline monooxygenase; BADH, betaine aldehyde dehydrogenase; CDH, choline dehydrogenase; Cho, choline; CMO, choline monooxygenase; COX, choline oxidase; GB, glycine betaine; GUS, β-glucuronidase; Hgr, hygromycin resistance gene; INH, isonicotinic acid hydrazide; Km, kanamycin resistance gene; NOS, nopaline synthase; Ter, terminator; TGA, root-specific auxin-responsive element.
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